|RFC standard||RFC 10|
|Source||pAAV_MCS: provided by Stratagene|
|Submitted by||FreiGEM 2010|
Producing recombinant virus particles for therapeutical applications is, besides specific cell targeting, purification and quantification assays of AAV-2, one intention of the Virus Construction Kit provided by the iGEM team Freiburg_Bioware 2010. For obtaining a modular toolkit, the complex biological system of the Adeno-associated virus serotype 2 was examined by an exhaustive literature search. Subsequently, the essential components for AAV-2 particle production were extracted and redesigned to match the iGEM standard.
The provided tripartite system is independent of a superinfection of Adeno- or herpes simplex viruses since the genes encoding the required helper-proteins are co-transfected. Inside the eukaryotic host cell, the DNA sequence containing the inverted terminal repeats (ITRs) is extracted and later encapsidated into the preformed capsids after production of single-stranded DNA. Consequently, this plasmid is known as the vector plasmid (pGOI). Promoter, beta-globin intron and the hGH terminator signal are flanked by the ITRs (ITRs, BBa_K404100 and BBa_K404101) and regulate transgene expression. The vector plasmid containing the desired gene of interest is cotransfected with the RepCap plasmid (BBa_K404001, BBa_K404002 or BBa_K404003) and the pHelper plasmid. To obtain the fully assembled vector plasmid, several assembly steps have to be performed.
Furthermore, by providing the tumor-specific promoter with the “Virus Vonstruction Kit”, the iGEM Freiburg_Bioware team 2010 ensures another layer of specificity and safety to the recombinant viral vector system. Telomerase activation is a critical step in human tumorigenesis and about 90% of human tumors show telomerase activity. In most somatic cells, the phTERT promoter is inactive. This prevents expression of the hTERT protein subunit and renders the healthy tissue telomerase negative.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25Illegal NgoMIV site found at 1122
Illegal AgeI site found at 1842
- 1000Illegal BsaI site found at 2244